The long term goal of this proposal is to elucidate the roles of intracellular calcium signals in circadian clock neurons. Experiments will be carried out in the fruit fly, Drosophila melanogaster, which has a functionally sophisticated and anatomically well- characterized circadian control system and is uniquely amenable to application of genetic methods that target clock neurons in the intact behaving animal. In addition to canonical transcriptional feedback mechanisms, circadian oscillation also relies upon depolarization-activated ionic membrane conductances. The proposed aims explore the hypothesis that intracellular calcium signals triggered by membrane depolarization are a core component of the cellular circadian oscillator. An engineered calcium buffer protein is specifically targeted to clock neurons in the brains of transgenic flies to disrupt cellular calcium signals in the intact living organism, followed by measurement of effects on circadian rhythms of locomotor activity, cellular accumulation of known transcription factor components of the circadian oscillator, and intracellular calcium dynamics. Preliminary studies using this approach indicate that intracellular calcium buffering in clock neurons leads to dose-dependent slowing of free-running behavioral and cellular rhythms with arrhythmicity at the highest dose. The proposed aims will identify the subcellular location of the relevant calcium signals, their temporal dynamics, the detailed effects their disruption has on cellular rhythms, and the downstream calcium-sensitive signaling pathways required for their transduction. Because of the great similarity in the genetic and cellular bases for circadian rhythmicity in flies and mammals, the information that can uniquely be obtained exploiting the genetic accessibility of Drosophila in the proposed studies will provide insight into general principles of cellular oscillator function that are relevant both to the basic circadian research done in mammalian model systems and to clinical research on human disorders of circadian function. Disruption of daily rhythms of rest and activity in human beings--through genetic mutation (as in advanced sleep phase disorder), disease, or environmental conditions (as in "jet lag" or for night shift workers)--has many adverse consequences for public health, workplace safety, and economic productivity. Understanding the cellular mechanisms of these rhythms is key to developing drugs and other treatments for their amelioration. The proposed studies will provide insight into general principles of cellular oscillator function that are relevant both to the basic circadian research done in mammalian model systems and to clinical research on human disorders of circadian function. Disruption of daily rhythms of rest and activity in human beings--through genetic mutation (as in advanced sleep phase disorder), disease, or environmental conditions (as in "jet lag" or for night shift workers)--has many adverse consequences for public health, workplace safety, and economic productivity. Understanding the cellular mechanisms of these rhythms is key to developing drugs and other treatments for their amelioration. [unreadable] [unreadable] [unreadable]